Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Semiautomated determination of minimum bactericidal concentrations of antibiotics

Using a newly devised 50-channel photometer which records the opacity of growing bacterial cultures, it was shown that the time taken by cultures diluted 1/1000 in fresh broth to reach 50% of the opacity of a fully grown culture was inversely related to the concentration of organisms in the original culture. This relation was used to determine the numbers of survivors after exposure to benzylpenicillin and gentamicin alone and in combination. The procedure is commended as a labour-saving and potentially rapid method of obtaining comprehensive information on the bactericidal action and interaction of antibiotics.     

Airway intervention in adult supraglottitis

Background The timing of aggressive airway intervention in adult epiglottitis is controversial. Aims To correlate Friedman’s staging of epiglottitis on admission with the airway interventions undertaken. Methods A retrospective study of 23 adult patients, mean age 51 years (range 29–81 years), who had been admitted with acute supraglottitis between March 1988 and December 2000 was undertaken. Results Three patients (13%) had airway interventions; two with tracheostomy and one with tracheal intubation. All were Friedman stage III and had rapid symptom progression during the 24 hours prior to admission. Three other stage III patients with symptom progression longer than 24 hours and all the remaining patients (stage II or less) were managed with observation and intravenous therapy. Conclusions Friedman originally advocated airway intervention in any patient stage II or worse, but this intubation threshold should probably be lowered to those patients with rapid-onset stage III (moderate respiratory distress, stridor, respiratory rate >30 per minute, pCO₂ >45mmHg) disease.     

Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection

We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.     

Rapid extraction of fungal DNA from clinical samples for PCR amplification.

A hexadecyltrimethylammonium bromide (CTAB) method for isolating fungal DNA from clinical samples, suitable for PCR amplification is described. Yeast and filamentous fungi DNA from clinical samples was amplified with primers complementary to the genes coding for rRNA, amplifying a 105 bp fragment and internal transcribed spacer primers amplifying fragments between 242 and 622 bp. The level of sensitivity was 10 +/- 5 yeast and 28 Aspergillus fumigatus CFU ml-1 of biological fluid.     

Abnormal vaginal flora in symptomatic non-pregnant and pregnant women in a Greek hospital: a prospective study

Bacterial vaginosis (BV), candidiasis, and trichomoniasis were the three established types of vaginal conditions until aerobic vaginitis (AV) was defined in the early 2000s. We sought to study the prevalence of abnormal vaginal flora (AVF) with inflammation in our hospital and to correlate it with AV. We prospectively collected vaginal smear specimens originated from symptomatic women who were examined at Iaso Obstetrics, Gynecology and Children’s Hospital of Athens from April 2014 until September 2015. Amsel’s criteria were used for the diagnosis of BV. The presence of leukocytes and lactobacillary grade were evaluated to classify a condition as AVF with inflammation; subsequently, bacterial cultures were performed. A total of 761 women were included. Five hundred and seventy-nine women were diagnosed with candidiasis, BV, trichomoniasis, or other types of vaginitis in which no pathogenic bacterial growth occurred in cultures. One hundred and eighty-two women (23.9 %) were diagnosed with AVF with inflammation (116 non-pregnant, 66 pregnant). Escherichia coli was the most common pathogen among these women (non-pregnant: 45.7 %, pregnant: 34.8 %). Other common pathogens were Group-B-Streptococcus (non-pregnant: 20.7 %, pregnant: 22.7 % respectively), Enterococcus faecalis (14.7 %, 18.2 %), and Klebsiella pneumoniae (6.9 %, 12.1 %). The prevalence of AVF with inflammation may be high. Since inflammation criteria were applied, most cases of BV were eliminated and the majority of cases of AVF are AV. Therefore, clinicians should include AV in the differential diagnosis of vaginitis, while microbiologists should take into account the growth of aerobic bacteria in vaginal cultures originating from women with microscopic findings of AV.     

Multiple clones within multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104. The Greek Nontyphoidal Salmonella Study Group

Six distinct clones were present among Greek multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3')-Ia, pse-1, and tem-1.